Method for selecting and amplifying polynucleotides

ABSTRACT

The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/786,198 filed Feb. 10, 2020 which is a continuation of U.S.application Ser. No. 15/489,549 filed Apr. 17, 2017 now U.S. Pat. No.10,597,653 issued Mar. 24, 2020 which is a continuation of U.S.application Ser. No. 14/611,991 filed Feb. 2, 2015 now U.S. Pat. No.9,624,489 issued Apr. 18, 2017 which is a continuation of U.S.application Ser. No. 13/387,078 filed Jan. 25, 2012 now U.S. Pat. No.8,999,642 issued Apr. 7, 2015 which is the U.S. National Phase of Int.App. No. PCT/US2009/054945 filed Aug. 25, 2009 which is acontinuation-in-part of U.S. application Ser. No. 12/395,299 filed Feb.27, 2009 now abandoned which claims priority to U.S. Prov. App. No.61/035,254 filed Mar. 10, 2008, which are each incorporated by referencein its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSEQLISTING ILLINC537C3, created Sep. 8, 2021, which is approximately 2Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The current invention relates to the field of nucleic acidamplification. More specifically, the present embodiments providemethods for selecting one or more regions of a nucleic sample on a solidsupport and growing nucleic acid clusters directly on the solid supportwhilst eliminating the need for multiple sample titration steps.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure in its entirety of each ofthese publications and documents is incorporated by reference herein.

A number of methods for high throughput nucleic acid sequencing rely ona universal amplification reaction, whereby a DNA sample is randomlyfragmented, then treated such that the ends of the different fragmentsall contain the same DNA sequence. Fragments with universal ends canthen be amplified in a single reaction with a single pair ofamplification oligonucleotides. Separation of the library of fragmentsto the single molecule level prior to amplification ensures that theamplified molecules form discrete populations that can then be furtheranalysed. Such separations can be performed either in emulsions, or on asurface. Alternatively it is possible to design amplificationoligonucleotides which are specific to certain portions of the nucleicacid sample, and hence remove the need to modify the ends of the sample.

Polynucleotide arrays have been formed based on ‘solid-phase’ nucleicacid amplification. For example, a bridging amplification reaction canbe used wherein a template immobilised on a solid support is amplifiedand the amplification products are formed on the solid support in orderto form arrays comprised of nucleic acid clusters or ‘colonies’. Eachcluster or colony on such an array is formed from a plurality ofidentical immobilized polynucleotide strands and a plurality ofidentical immobilised complementary polynucleotide strands. The arraysso formed are generally referred to herein as ‘clustered arrays.’

In common with several other amplification techniques, solid-phasebridging amplification uses forward and reverse amplificationoligonucleotides which include ‘template specific’ nucleotide sequenceswhich are capable of annealing to sequences in the template to beamplified, or the complement thereof, under the conditions of theannealing steps of the amplification reaction. The sequences in thetemplate to which the primers anneal under conditions of theamplification reaction may be referred to herein as ‘primer binding’sequences.

Certain embodiments of clustering methods make use of ‘universal’primers to amplify a variable template portion that is to be amplifiedand that is flanked 5′ and 3′ by common or ‘universal’ primer bindingsequences. The ‘universal’ forward and reverse primers include sequencescapable of annealing to the ‘universal’ primer binding sequences in thetemplate construct. The variable template portion, or ‘target’ mayitself be of known, unknown or partially known sequence. This approachhas the advantage that it is not necessary to design a specific pair ofprimers for each target sequence to be amplified; the same primers canbe used for amplification of different templates provided that eachtemplate is modified by addition of the same universal primer-bindingsequences to its 5′ and 3′ ends. The variable target sequence cantherefore be any DNA fragment of interest. An analogous approach can beused to amplify a mixture of templates (targets with known ends), suchas a plurality or library of target nucleic acid molecules (e.g.,genomic DNA fragments), using a single pair of universal forward andreverse primers, provided that each template molecule in the mixture ismodified by the addition of the same universal primer-binding sequences.

Such ‘universal primer’ approaches to PCR amplification, and inparticular solid-phase bridging amplification, are advantageous sincethey enable multiple template molecules of the same or different, knownor unknown sequence to be amplified in a single amplification reaction,which may be carried out on a solid support bearing a single pair of‘universal’ primers. Simultaneous amplification of a mixture oftemplates of different sequences can otherwise be carried out with aplurality of primer pairs, each pair being complementary to each uniquetemplate in the mixture. The generation of a plurality of primer pairsfor each individual template can be cumbersome and expensive for complexmixtures of templates. In certain applications such as detecting thepresence of a viral or microbial infection, or for characterising apopulation of microbes, it may be possible to design the amplificationoligonucleotides such that only the nucleic acid from the microbes isamplified.

In preparing a clustered array, typically the higher the concentrationof template used, the higher the density of clusters that will beproduced on a clustered array. If the density of clusters is too great,it may be difficult to individually resolve each cluster and overlappingcolonies may be formed. A titration can be performed to determine theoptimal template concentration to achieve an optimal cluster density onthe array wherein each cluster can be separately resolved. However, suchtitrations can lead to a loss of valuable flow cell channels due to acluster density that is too high or too low, a loss of template sample,an increase in the level of reagents required or an increase in sampleprocessing time.

Thus, there is a need for a method of controlling and achieving desiredcluster density that is independent of the concentration of the originalnucleic acid sample and avoids nucleic acid titration steps. The presentinvention satisfies this need and provides other advantages as well

SUMMARY OF THE INVENTION

The invention provides in certain embodiments a method of selecting andamplifying polynucleotides. The method can include (a) providing anucleic acid sample having a plurality of template polynucleotides; (b)providing a plurality of oligonucleotides immobilised on a solid supportwherein the plurality of oligonucleotides includes (i) a plurality ofcapture oligonucleotides each having a different sequence capable ofhybridising to a selected region of the nucleic acid sample, and (ii) aplurality of amplification oligonucleotides, wherein the captureoligonucleotides are immobilised at a lower density than theamplification oligonucleotides; (c) applying the templatepolynucleotides to the solid support under conditions such that thetemplate polynucleotides selectively hybridise to the captureoligonucleotides; (d) extending the capture oligonucleotides to generateextension products complementary to the template polynucleotides; and(e) amplifying the extension products using the one or moreamplification sequences immobilised on the solid support.

In a particular aspect, the invention provides a method of controllingthe sequence and density of colonies of amplified single strandedpolynucleotides formed on a solid support. The method can include thesteps of (a) providing a plurality of template polynucleotides; (b)providing a plurality of at least three oligonucleotides immobilised toa solid support wherein at least one of the oligonucleotides is acapture oligonucleotide capable of hybridising to the templatepolynucleotides, and at least two of the oligonucleotides areamplification oligonucleotides which are incapable of hybridising to thetemplate polynucleotides, wherein the capture oligonucleotides areimmobilised at a lower density than the amplification oligonucleotidesand the capture oligonucleotides are selective for a portion of theplurality of template polynucleotides; (c) applying the templatepolynucleotides to the solid support under suitable conditions such thatthe template polynucleotide molecules selectively hybridise to thecapture oligonucleotides; (d) extending the capture oligonucleotidesusing a nucleic acid polymerase to generate double stranded extensionproducts complementary to the single stranded template polynucleotides;(e) denaturing the double stranded extension products to remove thehybridised single stranded polynucleotide template molecules from theextension products to produce single stranded template moleculesimmobilised on the solid support; and (f) amplifying the single strandedtemplate molecules immobilised on the solid support using the two ormore amplification oligonucleotides immobilised on the solid support;wherein the density of the immobilised colonies is controlled by thedensity of the capture oligonucleotides rather than the concentration ofthe single stranded template polynucleotides.

The invention also provides in certain embodiments a flow cell uniformlygrafted with a plurality of oligonucleotides, wherein the pluralityincludes four species of oligonucleotides having different sequences,wherein two of the four species (e.g., a first and a second species) arepresent at a lower density than the other two species (e.g., a third anda fourth species).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method of the invention wherein the captureoligonucleotide is longer than the amplification oligonucleotides, andthe template selectively hybridises to the capture oligonucleotide thatextends beyond the amplification oligonucleotide. The captureoligonucleotide is extended opposite the template strand, and thetemplate strand is denatured and removed. The immobilised template copycan hybridise to one of the immobilised amplification oligonucleotides,and the amplification oligonucleotide can be extended. The captureoligonucleotide also comprises a sequence corresponding to one of theamplification oligonucleotides, and hence upon synthesising a duplexfrom the immobilised template copy, both ends of the immobilised duplexcan comprise sequences complementary to one of the amplificationoligonucleotides.

FIG. 2 shows an exemplary method of preparing a single stranded templatelibrary suitable for amplification.

FIG. 3 shows an exemplary method of the invention wherein one of theamplification oligonucleotides is initially blocked from strandelongation. After extending the immobilised template strand, the blockis removed and the sample can proceed through cycles of bridgeamplification.

FIG. 4 shows an exemplary solid support with two different species ofimmobilised amplification oligonucleotides and one species of captureoligonucleotide.

FIG. 5 shows a nucleic acid sample fragmented into a plurality ofpolynucleotides containing a selected target region. Upon fragmentation,some fragments contain the target region, thereby providing templatesfor subsequent capture, while other fragments do not contain a targetregion and can not therefore become templates. The fragments may undergoligation of an adapter at one end. The adapter may be complementary to,or the same as one of the amplification oligonucleotides on the support.

FIG. 6 shows hybridisation of a sample of template polynucleotides fromFIG. 5 to a support. The sample hybridises to the captureoligonucleotide via the target region, and the remaining molecules insample, which do not contain the target region do not hybridise and canbe washed from the support. The molecules captured on the support can beused as template polynucleotides.

FIG. 7 shows that the capture oligonucleotides that have captured thetemplate polynucleotides from FIG. 6 can be extended to make extensionproducts complementary to the template polynucleotides. The templatepolynucleotides can be denatured. If the templates carry an adaptersequence, the adapter sequence is copied as part of the extension. Ifthe copy of the adapter sequence is complementary to an amplificationoligonucleotide, the extension products can be amplified using theamplification oligonucleotides on the support.

FIG. 8 shows an assay for analysing a population of microbes using 16Sribosomal RNA sequencing. The capture oligonucleotides on the supportare shown as being selective for two of the constant regions of thebacterial 16S ribosomal RNA gene (8F and 553R). These two primers can beused to amplify approximately 500 base pairs of the approximately 1500base pair gene, and include the V1, V2 and V3 variable regions. Thecapture oligonucleotides are produced by extension of the P5 and P7amplification oligonucleotides. The capture oligonucleotides are thenused to specifically capture the fragments of the 16S rRNA genes fromthe sample. The capture oligonucleotides are then extended. Eachextended capture oligonucleotide can be turned into a cluster by solidphase amplification using the amplification oligonucleotides. Sequencingthe clusters gives information about the members of the population ofmicrobes due to the different 16S RNA regions captured and sequenced, aseach microbe has a characteristic 16S gene sequence.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in certain embodiments to methods for selectingand controlling the density of different molecular species derivatizedon a surface. In particular embodiments, the molecular species arenucleic acids having different sequences. The invention is particularlyuseful for controlling the density of nucleic acid clusters produced ona solid support. An advantage of the methods is the reduction or evenelimination of the need for multiple sample titration steps forcontrolling density of molecules on surfaces. Another advantage of theinvention is the ability to select a portion of the nucleic acid samplevia sequence selective hybridisation to the capture oligonucleotide.

The methods set forth herein can be used with those described in U.S.application Ser. No. 12/395,229 including, for example, methods ofcontrolling the density of clusters by using capture oligonucleotides ona solid support. In particular embodiments the methods set forth hereininclude the use of the capture oligonucleotides to select a subset ofthe nucleic acid sample, and hence control both the sequence of theclusters and the number, or density, of clusters on the support.

In embodiments wherein surfaces are derivatized with nucleic acids forsubsequent formation of amplified clusters, the density of the clusteron the support can be controlled by the density of one of theimmobilised primers used for capturing the template samples. The densityof primers on every chip can be controlled during manufacturing, simplyby the ratio of the capture oligonucleotides to the amplificationoligonucleotides, and hence the density of clusters can be independentof the concentration or dilution of the template sample. For example,conditions can be used where the template sample is in a molar excessrelative to primers, such that the density of clusters will besubstantially the same even if the concentration of template is furtherincreased. This concentration independence removes the need toaccurately measure the initial concentration of double strandedtemplate, and is independent of the accurate dilution of the sample. Thedensity of clusters on multiple chips can be made substantially uniformby controlling the ratio and concentration of capture oligonucleotidesto amplification oligonucleotides attached to the chip surface. Becauseprimers can typically be synthesized and manipulated under morecontrolled conditions than template samples that are derived fromdifferent biological sources, the methods set forth herein provideincreased reproducibility in creating cluster arrays. Further advantagesare provided by creating pools of primers in a desired ratio that can bereused for creating multiple cluster arrays having reproducible density.

In accordance with the methods set forth herein a plurality ofoligonucleotides can be immobilised to a solid support. The pluralitycan include different species of oligonucleotide molecule each having adifferent sequence. For example, a plurality of oligonucleotides caninclude at least two different species of oligonucleotides, at leastthree different species, at least four different species or more,wherein a first species has a different sequence than the other speciesin the plurality. It will be understood that different species ofoligonucleotide can share a common sequence so long as there is asequence difference between at least a portion of the different species.For example, as shown in FIG. 3, the two species identified as P5′ andP5′ HybBlocked share a common sequence but the P5′ HybBlocked specieshas an additional hairpin forming sequence not found in the P5′ species.

The term ‘ immobilised’ as used herein is intended to encompass director indirect attachment to a solid support via covalent or non-covalentbond(s). In certain embodiments of the invention, covalent attachmentmay be used, but generally all that is required is that the molecules(for example, nucleic acids) remain immobilised or attached to a supportunder conditions in which it is intended to use the support, for examplein applications requiring nucleic acid amplification and/or sequencing.Typically oligonucleotides to be used as capture oligonucleotides oramplification oligonucleotides are immobilized such that a 3′ end isavailable for enzymatic extension and at least a portion of the sequenceis capable of hybridizing to a complementary sequence. Immobilizationcan occur via hybridization to a surface attached oligonucleotide, inwhich case the immobilized oligonucleotide or polynucleotide may be inthe 3′-5′ orientation. Alternatively, immobilization can occur by meansother than base-pairing hybridization, such as the covalent attachmentset forth above.

The term ‘solid support’ as used herein refers to any insolublesubstrate or matrix to which molecules can be attached, such as forexample latex beads, dextran beads, polystyrene surfaces, polypropylenesurfaces, polyacrylamide gel, gold surfaces, glass surfaces and siliconwafers. The solid support may be a planar glass surface. The solidsupport may be mounted on the interior of a flow cell to allow theinteraction with solutions of various reagents.

In certain embodiments the solid support may comprise an inert substrateor matrix which has been ‘functionalised’, for example by theapplication of a layer or coating of an intermediate material comprisingreactive groups that permit covalent attachment to molecules such aspolynucleotides. By way of non-limiting example such supports mayinclude polyacrylamide hydrogel layers on an inert substrate such asglass. In such embodiments the molecules (for example, polynucleotides)may be directly covalently attached to the intermediate layer (forexample, a hydrogel) but the intermediate layer may itself benon-covalently attached to other layers of the substrate or matrix (forexample, a glass substrate). Covalent attachment to a solid support isto be interpreted accordingly as encompassing this type of arrangement.

Primer oligonucleotides' or Amplification oligonucleotides' areoligonucleotide sequences that are capable of annealing specifically toa single stranded polynucleotide sequence to be amplified underconditions encountered in a primer annealing step of an amplificationreaction. Generally, the terms ‘nucleic acid, ‘polynucleotide’ andOligonucleotide’ are used interchangeably herein. The different termsare not intended to denote any particular difference in size, sequence,or other property unless specifically indicated otherwise. For clarityof description the terms may be used to distinguish one species ofmolecule from another when describing a particular method or compositionthat includes several molecular species.

A polynucleotide sequence that is to be copied or amplified is generallyreferred to herein as a ‘template.’ A template can include primerbinding sites that flank a template sequence that is to be amplified. Atemplate hybridised to a capture oligonucleotide may contain bases whichextend beyond the 5′ end of the capture oligonucleotide in such a waythat not all of the template is amenable to extension. In particularembodiments, as set forth in further detail below, a plurality oftemplate polynucleotides includes different species that differ in theirtemplate sequences but have primer binding sites that are the same fortwo or more of the different species. The two primer binding sites whichmay flank a particular template sequence can have the same sequence,such as a palindromic sequence or homopolymeric sequence, or the twoprimer binding sites can have different sequences. Accordingly, aplurality of different template polynucleotides can have the same primerbinding sequence or two different primer binding sequences at each endof the template sequence. Thus, species in a plurality of templatepolynucleotides can include regions of known sequence that flank regionsof unknown sequence that are to be evaluated, for example, bysequencing. Template polynucleotides may carry a single adapter speciesto serve as a primer binding sequence at a single end only. In caseswhere the templates carry an adapter at a single end, this may be eitherthe 3′ end or the 5′ end. Template polynucleotides may be used withoutany adapter, in which case the primer binding sequence comes directlyfrom a sequence found in the nucleic acid sample.

Generally amplification reactions use at least two amplificationoligonucleotides, often denoted ‘forward’ and ‘reverse’ primers.Generally amplification oligonucleotides are single strandedpolynucleotide structures. They may also contain a mixture of natural ornon-natural bases and also natural and non-natural backbone linkages,provided, at least in some embodiments, that any non-naturalmodifications do not permanently or irreversibly preclude function as aprimer—that being defined as the ability to anneal to a templatepolynucleotide strand during conditions of an extension or amplificationreaction and to act as an initiation point for the synthesis of a newpolynucleotide strand complementary to the annealed template strand.That being said, in certain embodiments the present invention mayinvolve the use of a subset of primers, either forward or reverse, thathave been modified to preclude hybridisation to a templatepolynucleotide strand, the modification being altered or reversed atsome point such that hybridisation is no longer precluded.

Primers may additionally comprise non-nucleotide chemical modifications,for example to facilitate covalent attachment of the primer to a solidsupport. Certain chemical modifications may themselves improve thefunction of the molecule as a primer or may provide some other usefulfunctionality, such as providing a cleavage site that enables the primer(or an extended polynucleotide strand derived therefrom) to be cleavedfrom a solid support. Useful chemical modifications can also providereversible modifications that prevent hybridisation or extension of theprimer until the modification is removed or reversed. Similarly, othermolecules attached to a surface in accordance with the invention caninclude cleavable linker moieties and or reversible modifications thatalter a particular chemical activity of function of the molecule.

A plurality of oligonucleotides used in the methods set forth herein caninclude species that function as capture oligonucleotides. The captureoligonucleotides may include a ‘template specific portion’, namely asequence of nucleotides capable of annealing to a selected region of thenucleic acid sample in a polynucleotide molecule of interest such as onethat is to be amplified. The capture oligonucleotides may comprise asequence which is specific for a subset of the molecules in a nucleicacid sample. Thus only a subset of the molecules in the sample may inthese and related embodiments be selected by the captureoligonucleotides to become template polynucleotides. The captureoligonucleotides may comprise a single species of oligonucleotide, ormay comprise two or more species with a different sequence. Thus thecapture oligonucleotide may be two or more sequences, 10 or moresequences, 100 or more sequences, 1000 or more sequences or 10000 ormore sequences. The primer binding sequences will generally be of knownsequence and will therefore be complementary to a region of knownsequence of the single stranded polynucleotide molecule. The captureoligonucleotides may include a capture oligonucleotide and anamplification oligonucleotide. For example, as shown in FIG. 1, acapture oligonucleotide may be of greater length than amplificationoligonucleotides that are attached to the same substrate, in which casethe 5′ end of the capture oligonucleotides may comprise a region withthe same sequence as one of the amplification oligonucleotides. Aportion of a template, such as the 3′ end of the template, may becomplementary to the 3′ of the capture oligonucleotides. The 5′ end ofthe template may contain a region that comprises a sequence identical toone of the amplification oligonucleotides such that upon copying thetemplate, the copy can hybridise to the immobilized amplificationoligonucleotide. Thus, an oligonucleotide species that is useful in themethods set forth herein can have a capture oligonucleotide, anamplification oligonucleotide or both. Conversely, an oligonucleotidespecies can lack a capture oligonucleotide, an amplificationoligonucleotide or both. In this way the hybridization specificity of anoligonucleotide species can be tailored for a particular application ofthe methods.

The length of primer binding sequences need not be the same as those ofknown sequences of polynucleotide template molecules and may be shorterin certain embodiments, for example, being particularly 16-50nucleotides, more particularly 16-40 nucleotides and yet moreparticularly 20-30 nucleotides in length. The desired length of theprimer oligonucleotides will depend upon a number of factors. However,the primers are typically long (complex) enough so that the likelihoodof annealing to sequences other than the primer binding sequence is verylow. Accordingly, known sequences that flank a template sequence caninclude a primer binding portion and other portions such as a captureoligonucleotide, tag sequence or combination thereof.

‘Solid phase amplification,’ when used in reference to nucleic acids,refers to any nucleic acid amplification reaction carried out on or inassociation with a solid support. Typically, all or a portion of theamplified products are synthesised by extension of an immobilisedprimer. In particular the term encompasses solid phase amplificationreactions analogous to standard solution phase amplifications exceptthat at least one of the amplification oligonucleotides is immobilisedon the solid support.

As will be appreciated by the skilled reader, a given nucleic acidamplification reaction can be carried out with at least one type offorward primer and at least one type of reverse primer specific for thetemplate to be amplified. However, in certain embodiments, the forwardand reverse primers may include template specific portions of identicalsequence. In other words, it is possible to carry out solid phaseamplification using only one type of primer and such single primermethods are encompassed within the scope of the invention. The one typeof primer may include (a) subset (s) of modified primer (s) that havebeen modified to preclude hybridisation to a template polynucleotidestrand, the modification being removed, altered or reversed at somepoint such that hybridisation is no longer precluded. Other embodimentsmay use forward and reverse primers which contain identical templatespecific sequences but which differ in some structural features. Forexample, one type of primer may contain a non-nucleotide modificationwhich is not present in the other. In still yet another embodiment, thetemplate specific sequences are different and only one primer is used ina method of linear amplification. In other embodiments of the inventionthe forward and reverse primers may contain specific portions ofdifferent sequence.

In certain embodiments of the invention, amplification oligonucleotidesfor solid phase amplification are immobilised by covalent attachment tothe solid support at or near the 5′ end of the primer, such that aportion of the primer is free to anneal to its cognate template and the3′ hydroxyl group is free to function in primer extension. Again, incertain embodiments there is provided a subset of modified primers thatare prevented from hybridisation and/or extension until the modificationis removed, reversed or altered. In particular embodiments, theamplification oligonucleotides will be incapable of hybridisation to theinitial single stranded template. In such embodiments, hybridisation ofthe single stranded template will typically be specific for the captureoligonucleotides such that the amount of capture oligonucleotides on thesurface determines the amount of template captured and thus the densityof the resulting amplified clusters.

The chosen attachment chemistry will typically depend on the nature ofthe solid support and any functionalization or derivatization applied toit. In the case of nucleic acid embodiments, the primer itself mayinclude a moiety which may be a non-nucleotide chemical modification tofacilitate attachment. For example, the primer may include a sulphurcontaining nucleophile such as a phosphorothioate or thiophosphate atthe 5′ end. In the case of solid supported polyacrylamide hydrogels,this nucleophile may bind to a bromoacetamide group present in thehydrogel. In one embodiment, the means of attaching primers to the solidsupport is via 5′ phosphorothioate attachment to a hydrogel comprised ofpolymerized acrylamide and N-(5-bromoacetamidylpentyl) acrylamide(BRAPA).

A uniform, homogeneously distributed ‘lawn’ of immobilisedoligonucleotides may be formed by coupling (grafting) a solution ofoligonucleotide species onto the solid support. The solution can containa homogenous population of oligonucleotides but will typically contain amixture of different oligonucleotide species. The mixture can include,for example, at least two, three or more different species ofoligonucleotide. Each surface that is exposed to the solution thereforereacts with the solution to create a uniform density of immobilisedsequences over the whole of the exposed solid support. As such, aportion of the surface having a mixture of different immobilizedsequences can be surrounded by an area of the surface having a mixtureof the same immobilized sequences. A suitable density of amplificationoligonucleotides is at least 1 fmol/mm² (6×10¹⁰ per cm²), or moreoptimally at least 10 fmol/mm² (6×10¹¹ per cm²). The density of thecapture oligonucleotides can be controlled to give an optimum clusterdensity of 10⁶-10⁹ clusters per cm². The ratio of captureoligonucleotide species to the amplification oligonucleotide species canbe any desired value including, but not limited to at least 1:100,1:1000 or 1:100000 depending on the desired cluster density andbrightness. Similar densities or ratios of other molecular species canbe used in embodiments where molecules other than nucleic acids areattached to a surface.

Capture oligonucleotides may be deposited on the solid support at thesame time as the amplification oligonucleotides. Alternatively,especially in situations where the template polynucleotides do not carrysequences complementary to the amplification oligonucleotides, thecapture oligonucleotides may be produced using a solid support carryingonly amplification oligonucleotides by extending a portion of theamplification oligonucleotides using a copy of the captureoligonucleotides as a template. For example, a population ofoligonucleotide probes which contain a sequence complementary to one ofthe amplification oligonucleotides and a sequence which extends beyondthe amplification oligonucleotides may be prepared. This population ofoligonucleotides may be hybridised to the amplification oligonucleotideson the support at a sufficiently low density that only a portion of theamplification oligonucleotides on the support become hybridised. Forexample, the hybridised molecules might be individually resolvable suchthat the average distance between neighbouring molecules is large enoughthat the two molecules can be detected separately by optical microscopy.The portion of the amplification oligonucleotides with hybridisedmolecules may then undergo extension, e.g., using a polymerase andnucleoside triphosphates. This has the advantage that the captureoligonucleotides can be produced from a standard common solid supportcontaining only the amplification oligonucleotides, i.e., the same solidsupport can be prepared for use in all applications without needing tomanufacture a different support each time the sequence of the captureoligonucleotides is altered; and the capture oligonucleotides designedseparately and added to the support.

Previously, the density of attached single stranded polynucleotidemolecules and hence the density of clusters has been controlled byaltering the concentration of template polynucleotide molecules appliedto a support. By utilising a modified primer or capture oligonucleotideas set forth herein, the density of clusters on the amplified array canbe controlled without relying on careful titration of the startingconcentration of template polynucleotide strand applied to the solidsupport. This has the significant advantage that the methods need notrely on accurate concentration measurements and dilutions of thetemplate polynucleotide molecules, thereby leading to increasedreliability, reduction in dilution errors and a reduction in time andquantity of reagents required in downstream processes. For each solidsupport that contains too many or too few clusters, there is a reductionin the amount of data generated for an analysis of the clusters. Thiscan mean that generating the required depth of coverage of the samplemay require additional analytical runs that would not be required if thecluster density was optimal. Too many clusters gives optical saturationand an increase in overlap between two amplified molecules; too fewclusters gives undesirably high amounts of dark space that do notgenerate any data, thereby wasting reagents that are more efficientlyused with a densely populated surface.

In a particular embodiment, for each cluster, an immobilisedcomplementary copy of a single stranded polynucleotide template moleculeis attached to the solid support by a method of hybridisation and primerextension. Methods of hybridisation for formation of stable duplexesbetween complementary sequences by way of Watson-Crick base-pairing areknown in the art. The immobilised capture oligonucleotides can include aregion of sequence that is complementary to a region or templatespecific portion of the single stranded template polynucleotidemolecule. An extension reaction may then be carried out wherein thecapture oligonucleotide is extended by sequential addition ofnucleotides to generate a complementary copy of the single strandedpolynucleotide sequence attached to the solid support via the captureoligonucleotide. The single stranded polynucleotide sequence notimmobilised to the support may be separated from the complementarysequence under denaturing conditions and removed, for example bywashing.

The terms ‘separate’ and Separating,’ when used in reference to strandsof a nucleic acid, refer to the physical dissociation of the DNA basesthat interact within for example, a Watson-Crick DNA-duplex of thesingle stranded polynucleotide sequence and its complement. The termsalso refer to the physical separation of these strands. Thus, the termcan refer to the process of creating a situation wherein annealing ofanother primer oligonucleotide or polynucleotide sequence to one of thestrands of a duplex becomes possible. After the first extensionreaction, the duplex is immobilised through a single 5′ attachment, andhence strand separation can result in loss of one of the strands fromthe surface. In cases where both strands of the duplex are immobilised,separation of the strands means that the duplex is converted into twoimmobilised single strands.

In one aspect of the invention, one or more of the amplificationoligonucleotides can be modified to prevent hybridisation of a region ortemplate specific portion of the single stranded polynucleotidemolecule. Alternatively or additionally, one or more of theamplification oligonucleotides may be modified to prevent extension ofthe primer during one or more extension reactions, thus preventingcopying of the hybridised templates. These modifications can betemporary or permanent.

Generally, the capture oligonucleotides will include a region of thesame sequence as the plurality of amplification oligonucleotides. Oncethe 3′ end of the extended immobilised template copy has hybridised toone of the amplification oligonucleotides and been extended, theresulting duplex will be immobilised at both ends and all of the basesin the capture oligonucleotide sequence will have been copied. Thus thecapture oligonucleotide may include both the amplificationoligonucleotide sequence, plus a further sequence that is complementaryto the end or central region of the template. Typically the sequencecomplementary to the template will not be present in any of theamplification oligonucleotides. Alternatively, the amplificationoligonucleotides can contain the sequences complementary to thetemplates, but the amplification oligonucleotides can be reversiblyblocked to prevent hybridisation and/or extension during one or moreextension step, such as a first extension step in a particularamplification process.

According to one aspect of the invention, one or more of theamplification oligonucleotides may include a modification that acts as areversible block to either template hybridisation or extension or both.By way of non-limiting example, such modifications may be manifest asthe presence of an additional sequence of nucleotides that iscomplementary to the amplification oligonucleotide. This additionalsequence can be present in a portion of the amplificationoligonucleotide and thus acts as an intramolecular hairpin duplex, or a3′ blocking group that prevents extension of the primer. Alternatively,the additional sequence may be found on a separate oligonucleotide thathybridizes to the amplification oligonucleotide. A particular feature ofsuch a modification is that it can be removed, altered or reversed suchthat the functionality of the modified primer oligonucleotide isrestored and the primer is able to undergo hybridisation and extensionduring later steps of the methods. Among other examples, the blockinggroup may be a small chemical species such as a 3′ phosphate moiety thatcan be removed enzymatically, may be an abasic nucleotide such that the3′ end of the primer is not capable of hybridisation (and therebyextension), or may be a sequence of nucleotides that can be selectivelyexcised from the immobilised strands, for example, using restrictionendonucleases that selectively cleave particular sequences ordeglycosylases that selectively cleave oligonucleotides having exogenousbases such as uracil deoxyribonucleotides or 8-oxoguanine.

In one embodiment a plurality of three types of oligonucleotides (forexample comprising capture oligonucleotides, forward and reverseamplification oligonucleotides) are immobilised to a solid support.

Alternatively the three oligonucleotides may be forward amplification,blocked forward amplification and reverse amplification, where theunblocked forward primer acts as the capture oligonucleotide.

The nucleic acid sample may be double or single stranded. In order toobtain effective hybridisation, the double stranded sample may bedenatured to form single stranded polynucleotide molecules. The singlestranded polynucleotide molecules may have originated in single-strandedform, as DNA or RNA or may have originated in double-stranded DNA(dsDNA) form (e.g., genomic DNA fragments, PCR and amplificationproducts and the like). Thus a single stranded polynucleotide may be thesense or antisense strand of a polynucleotide duplex. Methods ofpreparation of single stranded polynucleotide molecules suitable for usein the method of the invention using standard techniques are well knownin the art. The precise sequence of the primary polynucleotide moleculesmay be known or unknown during different steps of the methods set forthherein. It will be understood that a double stranded polynucleotidemolecule can be hybridized to an immobilized capture oligonucleotide asexemplified herein for single stranded polynucleotide molecules, so longas a single stranded region of the double stranded polynucleotide isavailable and complementary to the capture oligonucleotide sequence.

An exemplary method for the isolation of one strand of a double strandedmolecular construct is shown in FIG. 2. A sample of unknown sequence maybe fragmented, and adapters attached to the ends of each fragment. Onestrand of the adapters may contain a moiety for surface immobilisation,for example a biotin that can be captured onto a streptavidin surface.The adapters may be mismatch adapters, for example as described incopending application US 2007/0128624, the contents of which areincorporated herein by reference in their entirety. Amplification of themismatch or forked adapters using a pair of amplificationoligonucleotides, one of which carries a biotin modification means thatone strand of each duplex carries a biotin modification. Immobilisationof the strands onto a streptavidin surface means that thenon-biotinylated strand can be eluted simply by denaturation/strandseparation. The eluted constructs will be in single stranded form andupon exposure to hybridisation conditions can be used to hybridiseagainst the immobilised capture oligonucleotides which can be extended.

In a particular embodiment, the single stranded polynucleotide moleculesare DNA molecules. More particularly, the single stranded polynucleotidemolecules represent genomic DNA molecules, or amplicons thereof, whichinclude both intron and exon sequence (coding sequence), as well asnon-coding regulatory sequences such as promoter and enhancer sequences.Still yet more particularly, the single stranded polynucleotidemolecules are human genomic DNA molecules, or amplicons thereof.

In a particular embodiment, the nucleic acid molecules may be isolatedfrom a biological sample that comprises a mixture of differentorganisms. For example, the sample may contain or include a mixture ofdifferent bacteria or viruses, such as may be present in cells, tissuesor fluids of an individual organism, which may in certain embodiments bea human or other vertebrate. In order to work out which microbes arepresent in the sample, the ‘microbiome’, regions of the sample specificto bacteria may be sequenced, for example the 16S ribosomal RNA generegion from the DNA sample. Thus the amplification oligonucleotides, orthe capture oligonucleotide may be selective for one of the constantregions found across the 16S rRNA gene region for all bacteria or the18S gene region common across different eukaryotes.

An embodiment of the method described herein may be used to select andform clusters from the bacterial 16S ribosomal gene of any bacteria.Suitable bacteria may include (but are not intended to be limited to)Acinetobacter baumannii, Actinomyces odontolyticus, Bacillus cereus,Bacteroides vulgatus, Clostridium beijerinckii, Deinococcus radiodurans,Enterococcus faecalis, Escherichia coli, Helicobacter pylori,Lactobacillus gasseri, Listeria monocytogenes, Methanobrevibactersmithii, Neisseria meningitides, Propionibacterium acnes, Pseudomonasaeruginosa, Rhodobacter sphaeroides, Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcusmutans and Streptococcus pneumoniae.

The sample, for example a sample obtained from human gut, stool, salivaor skin, may be treated to extract the nucleic acid present in thesample. The total nucleic acid extracted from the sample may undergofragmentation and may be contacted with a solid support carryingamplification and capture oligonucleotides as described herein. Ifindividual capture oligonucleotides carry a region of sequence that iscomplementary to a gene sequence shared between all bacteria, then thebacterial nucleic acids will be captured, and the other nucleic acids,for example viral or human nucleic acids will not. The bacterial nucleicacids can then be amplified to form clusters. Variable regions of thecaptured nucleic acids can be detected, for example, by sequencing. Thesequence of the variable regions provide information that can be used toidentify the bacteria from which they were obtained. Upon sequencing theclusters, the ratio between the numbers of two or more bacteria in asample may be calculated by counting the number of times a particularsequence read is obtained across the millions of clusters on the solidsupport.

Specific amplification of the bacterial sample is possible if theamplification oligonucleotides are only complementary to the bacterialnucleic acid. The capture oligonucleotides may be modified to selectnucleic acids from a particular bacteria or virus. Multiple differentcapture oligonucleotides may be used in order to optimise selection ofnucleic acid from the desired organism.

Capture oligonucleotides for selecting 16S gene regions may contain thefollowing sequences:

SEQ Name Region 5′-3′ ID NO: 8F Before V1 AGAGTTTGATCCTGGCTCAG 1 1542RAfter V9 AAGGAGGTGATCCAGCCGCA 2 338F Before V3 ACTCCTACGGGAGGCAGCAG 3533R After V3 TTACCGCGGCTGCTGGCAC 4 967F Before V6 MWACGCGARRAACCTTACC 51046R After V6 CGACARCCATGCASCACCT 6

Wherein M, W, R and S are the standard degenerate base codes (M=A and/orC, W=A and/or T, R=G and/or A, and S=G and/or C).

The capture oligonucleotides may be attached directly to theamplification oligonucleotides, for example by preparingoligonucleotides containing both the amplification and captureoligonucleotides in a single construct and attaching this to a solidsupport. Alternatively the capture oligonucleotides may be prepared byattaching amplification oligonucleotides to a support and hybridising anoligonucleotide with a sequence complementary to the sequence of thecapture oligonucleotide and the sequence of the amplificationoligonucleotide to the amplification oligonucleotide. The complementaryoligonucleotides can act as templates for the preparation of the captureoligonucleotides by extension of the amplification oligonucleotides.

In a particular embodiment, a single stranded target polynucleotidemolecule has two regions of known sequence. Yet more particularly, theregions of known sequence will be at the 5′ and 3′ termini of the singlestranded polynucleotide molecule such that the single strandedpolynucleotide molecule will be of the structure:

-   -   5′ [known sequence I]-[target polynucleotide sequence]-[known        sequence II]-3′

Typically ‘known sequence I’ and ‘known sequence II’ will comprise morethan 20, or more than 40, or more than 50, or more than 100, or morethan 300 consecutive nucleotides. The precise length of the twosequences may or may not be identical. The primer binding sequencesgenerally will be of known sequence and will therefore particularly becomplementary to a sequence within known sequence I and known sequenceII of the single stranded polynucleotide molecule. The length of theprimer binding sequences need not be the same as those of known sequenceI or II, and may be shorter, being particularly 16-50 nucleotides, moreparticularly 16-40 nucleotides and yet more particularly 20-30nucleotides in length. Known sequence I can be the same as knownsequence II or the two can be different.

Methods of hybridisation for formation of stable duplexes betweencomplementary sequences by way of Watson-Crick base pairing are known inthe art. A region or part of the single stranded polynucleotide templatemolecules can be complementary to at least a part of the immobilisedcapture oligonucleotide oligonucleotides. The plurality ofpolynucleotides from the sample which do not act as templates due tonon-hybridisation with the capture oligonucleotides may be removed fromthe solid support, for example by washing or other form of fluid flow.Since the amplification oligonucleotides are either modified to preventhybridisation and/or extension, or are non-complementary to the templatestrands, only the capture oligonucleotides will be capable ofhybridisation and extension. An extension reaction may then be carriedout wherein the capture oligonucleotide is extended by sequentialaddition of nucleotides to generate an extension product which is acomplementary copy of the single stranded template polynucleotideattached to the solid support via the capture oligonucleotide. Thesingle stranded template polynucleotide sequence not immobilised to thesupport may be separated from the complementary sequence underdenaturing conditions and removed, for example by washing. The distancebetween the individual capture oligonucleotide on the surface thereforecontrols the density of the single stranded template polynucleotides andhence the density of clusters formed later on the surface is alsocontrolled.

In embodiments such as that shown in FIG. 3 wherein the modified forwardprimer oligonucleotides are blocked and are unable to be extended,generally all of the amplification oligonucleotides will hybridise tothe single stranded template polynucleotides. When the extensionreaction is carried out only the unmodified forward captureoligonucleotides are extended by sequential addition of nucleotides togenerate a complementary copy of the single stranded templatepolynucleotide attached to the solid support via the unmodified forwardprimer oligonucleotide. The single stranded template polynucleotidesequences not hybridised to the support may be separated from theun-extended blocked forward primer oligonucleotides under denaturingconditions and removed, for example by washing with a chemicaldenaturant such as formamide. The distance between the individualunmodified forward primer oligonucleotides on the surface thereforecontrols the density of the single stranded template polynucleotides andhence the density of clusters formed later on the surface is alsocontrolled.

Following the attachment of the complementary single stranded templatepolynucleotides, the modified/blocked primers can be treated to reverse,remove or alter the modification such that they become functionallyequivalent to the unmodified forward primer oligonucleotides. Forexample, the double stranded structure may be removed either bydenaturation, for example by heating or treatment with an alkalinesolution when it is formed by a separate hybridised polynucleotide.Alternatively, where the hybridised polynucleotide is covalently linked,enzymatic digestion could be used to sequence-selectively cleave thestrand, followed by denaturation. Such methods for removing the doublestranded structure are known in the art and would be apparent to theskilled person (Sambrook and Russell, Molecular Cloning, A LaboratoryManual, third edition, Cold Spring Harbor Laboratory Press (2001)).

In one embodiment of the invention, the single stranded templatepolynucleotide molecule can be attached to the solid support by ligationto double stranded primers immobilised to the solid support usingligation methods known in the art (Sambrook and Russell, supra). Suchmethods utilise ligase enzymes such as DNA ligase to effect or catalysethe joining of the ends of the two polynucleotide strands, in this case,the single stranded template polynucleotide molecule and the primeroligonucleotide ligate such that covalent linkages are formed. In thiscontext ‘joining’ means covalent linkage of two polynucleotide strandsthat were not previously covalently linked. Thus, an aim of certainembodiments of the invention can also be achieved by modifying the 3′end of a subset of primer oligonucleotides such that they are unable toligate to the single stranded template polynucleotides. By way ofnon-limiting example, the addition of 2′3′dideoxy AMP (dideoxyAMP) bythe enzyme terminal deoxynucleotidyl transferase (TdT) effectivelyprevents T4 DNA ligase from ligating treated molecules together.

An alternative method would be to have the capture oligonucleotides asduplex strands and the amplification oligonucleotides as single strands.Upon ligation of the single strands to the capture duplexes (which wouldbe the only immobilised species carrying a free 5′ phosphate) the 3′ endof the immobilised strand can be extended as described above. Upondenaturation of the hybridised template sequence, amplification of theimmobilised strand can proceed as described. Other such methods forattaching single strands will be apparent to those skilled in the art.

In a next step according to particular embodiments of the presentinvention, suitable conditions are applied to the immobilised singlestranded polynucleotide molecule and the plurality of amplificationoligonucleotides such that the single stranded polynucleotide moleculehybridises to an amplification oligonucleotide to form a complex in theform of a bridge structure. Suitable conditions such as neutralisingand/or hybridising buffers are well known in the art (See Sambrook etal., supra; Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1998)). The neutralising and/orhybridising buffer may then be removed.

Next by applying suitable conditions for extension an extension reactionis performed. The amplification oligonucleotide of the complex isextended by sequential addition of nucleotides to generate an extensionproduct complementary to the single stranded polynucleotide molecule.The resulting duplex is immobilised at both 5′ ends such that eachstrand is immobilised.

Suitable conditions such as extension buffers/solutions comprising anenzyme with polymerase activity are well known in the art (See Sambrooket al., supra; Ausubel et al. supra). In a particular embodiment dNTP'smay be included in the extension buffer. In a further embodiment dNTP'scould be added prior to the extension buffer. This bridge amplificationtechnique can be carried out as described, for example, in U.S. Pat. No.7,115,400 and US 2005/0100900 A1, the contents of which are incorporatedherein by reference.

Examples of enzymes with polymerase activity which can be used in thepresent invention are DNA polymerase (Klenow fragment, T4 DNApolymerase), heat-stable DNA polymerases from a variety of thermostablebacteria (such as Taq, VENT, Pfu, or TfI DNA polymerases) as well astheir genetically modified derivatives (TaqGold, VENTexo, or Pfu exo). Acombination of RNA polymerase and reverse transcriptase can also be usedto generate the extension products. Particularly the enzyme may in theseand related embodiments have strand displacement activity, moreparticularly the enzyme may be active at a pH of about 7 to about 9,particularly pH 7.9 to pH 8.8, yet more particularly the enzymes are incertain exemplary embodiments B st or Klenow.

The nucleoside triphosphate molecules used are typicallydeoxyribonucleotide triphosphates, for example dATP, dTTP, dCTP, dGTP,or are ribonucleoside triphosphates for example ATP, UTP, CTP, GTP. Thenucleoside triphosphate molecules may be naturally or non-naturallyoccurring.

After the hybridisation and extension steps, the support and attachednucleic acids can be subjected to denaturation conditions. A flow cellcan be used such that, the extension buffer is generally removed by theinflux of the denaturing buffer. Suitable denaturing buffers are wellknown in the art (See Sambrook et al., supra; Ausubel et al. supra). Byway of example it is known that alterations in pH and low ionic strengthsolutions can denature nucleic acids at substantially isothermaltemperatures. Formamide and urea form new hydrogen bonds with the basesof nucleic acids disrupting hydrogen bonds that lead to Watson-Crickbase pairing. In a particular embodiment the concentration of formamideis 50% or more. These result in single stranded nucleic acid molecules.If desired, the strands may be separated by treatment with a solution ofvery low salt (for example less than 0.01 M cationic conditions) andhigh pH (>12) or by using a chaotropic salt (e.g. guanidiniumhydrochloride). In a particular embodiment a strong base is used. Astrong base is a basic chemical compound that is able to deprotonatevery weak acids in an acid base reaction. The strength of a base isindicated by its pKb value, compounds with a pKb value of less thanabout 1 are called strong bases and are well known to one skilled in theart. In a particular embodiment the strong base is Sodium Hydroxide(NaOH) solution used at a concentration of from 0.05 M to 0.25 M,particularly 0.1 M.

Following the hybridization, extension and denaturation stepsexemplified above, two immobilised nucleic acids will be present, thefirst containing a sequence the same as the first template singlestranded polynucleotide molecule (that was initially immobilised) andthe second being a nucleic acid complementary thereto, extending fromone of the immobilised capture oligonucleotides. Both the immobilisedstrands are then able to initiate further rounds of amplification bysubjecting the support to further cycles of hybridisation, extension anddenaturation. Thus the amplification proceeds from a single strand to aduplex, one duplex to two duplexes, two duplexes to four duplexes etc.throughout the cycles of annealing, extension and denaturation.

It may be advantageous to perform optional washing steps in between eachstep of the amplification method. For example an extension bufferwithout polymerase enzyme with or without dNTPs could be applied to thesolid support before being removed and replaced with the full extensionbuffer.

Such further rounds of amplification can be used to produce a nucleicacid colony or ‘cluster’ comprising multiple immobilised copies of thesingle stranded polynucleotide sequence and its complementary sequence.

The initial immobilisation of the template polynucleotide molecule meansthat the extension product can hybridise with amplificationoligonucleotides located at a distance within the total length of thetemplate polynucleotide molecule. Other surface bound primers that areout of reach will not hybridize to the extension product. Thus theboundary of the nucleic acid colony or cluster formed is limited to arelatively local area surrounding the location in which the initialtemplate polynucleotide molecule was immobilised.

Once more copies of the polynucleotide extension products molecule andits complement have been synthesised by carrying out further rounds ofamplification, i.e. further rounds of hybridisation, extension anddenaturation, then the boundary of the nucleic acid colony or clusterbeing generated will be able to be extended further, although theboundary of the colony formed is still limited to a relatively localarea around the location in which the initial single strandedpolynucleotide molecule was immobilised. For example the size of eachamplified cluster may be 0.5-5 microns, and can be controlled by thenumber of cycles performed.

It can thus be seen that the method of the present invention allows thegeneration of a plurality of nucleic acid colonies from multiple singleimmobilised single stranded polynucleotide molecules and that thedensity of these colonies can be controlled by altering the proportionsof modified capture/amplification oligonucleotides used to graft thesurface of the solid support.

In one embodiment, the hybridisation, extension and denaturation stepsare all carried out at the same, substantially isothermal temperature.For example the temperature is from 37° C. to about 75° C., particularlyfrom 50° C. to 70° C., yet more particularly from 60° C. to 65° C. In aparticular embodiment the substantially isothermal temperature may bethe optimal temperature for the desired polymerase.

In a particular aspect, the method according to the first aspect of theinvention is used to prepare clustered arrays of nucleic acid colonies,analogous to those described in U.S. Pat. No. 7,115,400, US 2005/0100900A1, WO 00/18957 and WO 98/44151 (the contents of which are hereinincorporated by reference), by solid-phase amplification.

In yet another aspect more than one capture oligonucleotides and morethan two amplification oligonucleotides, for example, at least three orfour or more, different amplification oligonucleotide sequences may begrafted to the solid support. In this manner more than one library, withcommon sequences which differ between the libraries, could be utilisedto prepare clusters, such as, for example libraries prepared from twodifferent patients. Alternatively different selected regions could beamplified simultaneously by using different amplificationoligonucleotides. Whilst the clusters may overlap in space, they wouldbe able to be sequenced one after the other due to the differencesbetween the ends of the templates. For example, two different samplescan be captured using two different capture oligonucleotides. These canbe amplified from the same two amplification oligonucleotides. Thesamples can be differentiated due to the two different captureoligonucleotides, which can be used as the sites for hybridisation oftwo different sequencing primers. The use of different captureoligonucleotides thereby gives rise to a method of sample indexing usingdifferent sequencing primers.

Clustered arrays formed by the methods of the invention are suitable foruse in applications usually carried out on ordered arrays such asmicro-arrays. Such applications by way of non-limiting example includehybridisation analysis, gene expression analysis, protein bindinganalysis, sequencing, genotyping, nucleic acid methylation analysis andthe like. The clustered array may be sequenced before being used fordownstream applications such as, for example, hybridisation withfluorescent RNA or binding studies using fluorescent labelled proteins.

Sequencing Methods

The invention also encompasses methods of sequencing amplified nucleicacids generated by solid-phase amplification. Thus, the inventionprovides a method of nucleic acid sequencing comprising amplifying apool of nucleic acid templates using solid-phase amplification asdescribed above and carrying out a nucleic acid sequencing reaction todetermine the sequence of the whole or a part of at least one amplifiednucleic acid strand produced in the solid-phase amplification reaction.

Sequencing can be carried out using any suitable sequencing technique. Aparticularly useful method is one wherein nucleotides are addedsuccessively to a free 3′ hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of thenucleotide added may be determined after each nucleotide addition or atthe end of the sequencing process. Sequencing techniques usingsequencing by ligation, wherein not every contiguous base is sequenced,and techniques such as massively parallel signature sequencing (MPSS)where bases are removed from, rather than added to the strands on thesurface are also within the scope of the invention.

The initiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of the solid-phaseamplification reaction. In this connection, one or both of the adaptorsadded during formation of the template library may include a nucleotidesequence which permits annealing of a sequencing primer to amplifiedproducts derived by whole genome or solid-phase amplification of thetemplate library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification oligonucleotides are covalently immobilised onthe solid surface are so-called ‘bridged’ structures formed by annealingof pairs of immobilised polynucleotide strands and immobilisedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for typical nucleic acid sequencing techniques,since hybridisation of a conventional sequencing primer to one of theimmobilised strands is not favoured compared to annealing of this strandto its immobilised complementary strand under standard conditions forhybridisation.

In order to provide more suitable templates for nucleic acid sequencing,it may be advantageous to remove or displace substantially all or atleast a portion of one of the immobilised strands in the ‘bridged’structure in order to generate a template which is at least partiallysingle-stranded. The portion of the template which is single-strandedwill thus be available for hybridisation to a sequencing primer. Theprocess of removing all or a portion of one immobilised strand in a‘bridged’ double-stranded nucleic acid structure may be referred toherein as linearization’, and is described in further detail inWO07010251 and US20090118128, the contents of which are incorporatedherein by reference in their entirety.

Bridged template structures may be linearized by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g., cleavage of a diol linkage withperiodate), cleavage of abasic sites by cleavage with endonuclease (forexample ‘USER’, as supplied by NEB, Ipswich, Mass., USA, part numberM5505S), or by exposure to heat or alkali, cleavage of ribonucleotidesincorporated into amplification products otherwise comprised ofdeoxyribonucleotides, photochemical cleavage or cleavage of a peptidelinker.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion (s) of the cleaved strand (s)that are not attached to the solid support. Suitable denaturingconditions, for example sodium hydroxide solution, formamide solution orheat, will be apparent to the skilled reader with reference to standardmolecular biology protocols (Sambrook et al., supra; Ausubel et al.supra). Denaturation results in the production of a sequencing templatewhich is partially or substantially single-stranded. A sequencingreaction may then be initiated by hybridisation of a sequencing primerto the single-stranded portion of the template.

Thus, the invention encompasses methods wherein the nucleic acidsequencing reaction comprises hybridising a sequencing primer to asingle-stranded region of a linearized amplification product,sequentially incorporating one or more nucleotides into a polynucleotidestrand complementary to the region of amplified template strand to besequenced, identifying the base present in one or more of theincorporated nucleotide (s) and thereby determining the sequence of aregion of the template strand.

One sequencing method which can be used in accordance with the inventionrelies on the use of modified nucleotides having removable 3′ blocks,for example as described in WO04018497, US 2007/0166705A1 and U.S. Pat.No. 7,057,026, the contents of which are incorporated herein byreference in their entirety. Once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′—OH groupavailable to direct further sequence extension and therefore thepolymerase can not add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides, it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas a different label attached thereto, known to correspond to theparticular base, to facilitate discrimination between the bases addedduring each incorporation step. Alternatively, a separate reaction maybe carried out containing each of the modified nucleotides separately.

The modified nucleotides may carry a label to facilitate theirdetection. A fluorescent label, for example, may be used for detectionof modified nucleotides. Each nucleotide type may thus carry a differentfluorescent label, for example, as described in U.S. ProvisionalApplication No. 60/801,270 (Novel dyes and the use of their labelledconjugates), published as WO07135368, the contents of which areincorporated herein by reference in their entirety. The detectable labelneed not, however, be a fluorescent label. Any label can be used whichallows the detection of an incorporated nucleotide.

One method for detecting fluorescently labelled nucleotides comprisesusing laser light of a wavelength specific for the labelled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means. Suitable instrumentation for recordingimages of clustered arrays is described in U.S. Provisional ApplicationNo. 60/788,248 (Systems and devices for sequence by synthesis analysis),published as WO07123744, the contents of which are incorporated hereinby reference in their entirety. The invention is not intended to belimited to use of the sequencing method outlined above, as essentiallyany sequencing methodology which relies on successive incorporation ofnucleotides into a polynucleotide chain can be used. Suitablealternative techniques include, for example, Pyrosequencing™, FISSEQ(fluorescent in situ sequencing), MPSS and sequencing by ligation-basedmethods, for example as described in U.S. Pat. No. 6,306,597 which isincorporated herein by reference.

The nucleic acid sample may be further analysed to obtain a second readfrom the opposite end of the fragment. Methodology for sequencing bothends of a cluster are described in co-pending applications WO07010252,PCTGB2007/003798 and US 20090088327, the contents of which areincorporated by reference herein in their entirety. In one example, theseries of steps may be performed as follows; generate clusters,linearize, hybridise first sequencing primer and obtain first sequencingread. The first sequencing primer can be removed, and in the cases wherea tag sequence is present in the cluster, a second primer hybridised andthe tag sequenced. The nucleic acid strand may then be ‘inverted’ on thesurface by synthesising a complementary copy from the remainingimmobilised primers used in cluster amplification. This process ofstrand resynthesis regenerates the double stranded cluster. The originaltemplate strand can be removed, to linearize the resynthesized strandthat can then be annealed to a sequencing primer and sequenced in asecond or third sequencing run.

In the cases where strand resynthesis is employed, both strands can beimmobilised to the surface in a way that allows subsequent release of aportion of the immobilised strand. This can be achieved through a numberof mechanisms as described in WO07010251 and US20090118128, the contentsof which are incorporated herein by reference in their entirety. Forexample, one primer can contain a uracil nucleotide, which means thatthe strand can be cleaved at the uracil base using the enzymes uracilglycosylase (UDG) which removes the nucleoside base, and endonucleaseVIII that excises the abasic nucleotide. This enzyme combination isavailable as USER™ from New England Biolabs (NEB, Ipswich, Mass., USA,part number M5505). The second primer may comprise an 8-oxoguaninenucleotide, which is then cleavable by the enzyme FPG (NEB part numberM0240). This design of primers gives control of which primer is cleavedat which point in the process, and also where in the cluster thecleavage occurs. The primers may also be chemically modified, forexample with a disulfide or diol modification that allows chemicalcleavage at specific locations.

Flow Cells

The invention also relates to flow cells for the preparation ofamplified arrays of nucleic acids wherein the flow cells contain auniform coating of three, four or more immobilised primers. Thus asubstrate described herein can occur within or as a part of a flow celland the methods set forth herein can be carried out in a flow cell. Incontrast to spotted arrays of multiple sequences, the three, four ormore oligonucleotides can be coated over the whole of the array surfacerather than in discreet locations that comprise different sequences ineach small location. The arrays may be of a size of 1 cm2 or greaterwhereby the whole 1 cm2 or greater comprises a homogeneous coating ofmultiple copies of the same three, four or more sequences. A flow cellcan be distinguished from a ‘spotted array’ or photolithographicallysynthesised array due to the fact that the oligonucleotides are attachedto each and every surface; top, bottom, walls and ends of the flow cellchamber, rather than being an array that is mounted in a housing.However, if desired a flow cell that is used in a method set forthherein can have surfaces with different reactivity for oligonucleotidessuch that the oligonucleotides are only attached to one or a subset ofthe aforementioned surfaces or even to only a subset of regions withinthese surfaces.

The flow cell may in certain embodiments be coated with threeoligonucleotide species of different sequence composition, namely twoamplification oligonucleotides and a capture oligonucleotide. The flowcell may in certain embodiments be coated with no more than the threeoligonucleotide species. However in other particular embodiments, theflow cell can further include one or more other oligonucleotide specieswhether an amplification oligonucleotide, capture oligonucleotide, orother species of oligonucleotide. The capture oligonucleotide may bepresent at a lower concentration than the amplification oligonucleotide,for example at least 100, 1000 or 100,000 fold lower relativeconcentration. The two amplification oligonucleotides may be present atsimilar ratios to each other, for example varying by less than a factorof two. The capture oligonucleotides may be longer than theamplification oligonucleotides, and may comprise the amplificationoligonucleotide sequence region plus a capture oligonucleotide region,as shown for example in FIG. 1. Alternatively or additionally, theamplification oligonucleotides may be blocked to prevent hybridisationand/or extension. The sequence of the capture oligonucleotides may bedifferent between different capture oligonucleotides. In certain relatedbut distinct embodiments the flow cell may be coated with at least fourspecies of oligonucleotides having different sequences, wherein at leasta first and a second of the four species are present at a lower densitythan the third and fourth of the four species. For example, the firstand second species may be capture oligonucleotides and the third andfourth species may be amplification oligonucleotides. Thus, in the abovedescribed embodiments and in other related embodiments that arecontemplated, a solid support may carry two or more captureoligonucleotides of different sequences. The sequence of the captureoligonucleotides can allow for selection of a known portion of thenucleic acid sample. The capture sequences may be produced by extendingsome or all of the amplification sequences.

Although the invention has been exemplified herein for embodiments usingnucleic acid species, it will be understood that the same principles canbe applied to other molecular species. For example, surfaces ofsubstrates can be derivatized with other synthetic molecules such aspeptides, small molecule ligands, saccharides or the like. Bycontrolling the amount of different species of such molecules in thederivatization step, a desired density of each species can result.Samples of molecules that bind to one or more of these solid phasemolecules can be used without the need for titrating the samples becausethe density of molecules from the sample that bind to the surfaces willbe controlled by the density of their binding partners on the surface.Accordingly, attachment of molecules from the sample can be controlledthermodynamically in a process that is allowed to proceed to equilibriumas opposed to a kinetic process that requires more precise control ofreaction conditions and incubation times. Once bound to the surface themolecules from the sample can be subsequently modified or detected. Insuch embodiments, the surface can include reversibly modified syntheticmolecules such that altering or removing the modification can allow themolecules from the sample to be modified or detected for a particularanalytical assay or step.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1.-21. (canceled)
 21. A method of controlling for different amounts oftemplate nucleic acids in different samples in solid phase amplificationreactions, comprising: (a) obtaining a first flow cell comprising aplurality of oligonucleotides immobilized on the first flow cell,wherein the plurality of oligonucleotides comprises a plurality ofcapture probes and a plurality of amplification primers, wherein thecapture probes are immobilized on the first flow cell at a density lowerthan the amplification primers; (b) selectively hybridizing firsttemplate nucleic acids to the capture probes; (c) extending the captureprobes hybridized to the first template nucleic acids to obtain extendedpolynucleotides; (d) removing the first template nucleic acids from theextended polynucleotides; (e) amplifying the extended polynucleotides byhybridizing the extended polynucleotides to the amplification primers toobtain amplified first template nucleic acids; and (f) repeating steps(a)-(e) with a second flow cell and second template nucleic acids,wherein an amount of the capture probes on the first flow cell issubstantially the same as an amount of the capture probes on the secondflow cell, and wherein an amount of the amplified first template nucleicacids is substantially the same as an amount of the amplified secondtemplate nucleic acids.
 22. The method of claim 21, wherein theamplification primers are blocked prior to step (d); and wherein step(d) further comprises deblocking the amplification primers.
 23. Themethod of claim 22, wherein the amplification primers are blocked fromextension by a chemical species attached to a 3′ end of theamplification primers.
 24. The method of claim 23, wherein the chemicalspecies is a phosphate group.
 25. The method of claim 21, wherein theamplifying comprises bridge amplification.
 26. The method of claim 21,wherein the amplifying comprises isothermal amplification.
 27. Themethod of claim 21, wherein the capture probes are immobilised on thefirst flow cell at a density of at least 2-fold lower than theamplification primers.
 28. The method of claim 27, wherein the captureprobes are immobilised on the first flow cell at a density of at least100-fold lower than the amplification primers.
 29. The method of claim21, wherein the capture probes are longer than the amplificationprimers.
 30. The method of claim 29, wherein capture probes comprise thesequence of the amplification primers.
 31. The method of claim 21,wherein the first template nucleic acids are different from one another.32. The method of claim 21, wherein at least one end of the first andsecond template nucleic acids comprises an adapter sequence.
 33. Themethod of claim 32, wherein the first template nucleic acids comprisethe same adapter sequences.
 34. The method of claim 32, wherein theamplification primers comprise a nucleotide sequence capable ofhybridizing to the adapter sequence or a complement thereof.
 35. Themethod of claim 21 wherein the first template nucleic acids are obtainedfrom a population of organisms.
 36. The method of claim 21, wherein thefirst template nucleic acids comprise genomic DNA.
 37. The method ofclaim 21, further comprising obtaining the first template nucleic acidsby ligating adapters to ends of a plurality of nucleic acid fragments.38. The method of claim 21, further comprising sequencing the amplifiedfirst template nucleic acids.
 39. A method of controlling for differentamounts of template nucleic acids in different samples in solid phaseamplification reactions, comprising: (a) performing a first selectiveamplification on a first flow cell with first template nucleic acids toobtain an amount of amplified first template nucleic acids, comprising:(i) obtaining the first flow cell comprising a first plurality ofoligonucleotides immobilized thereon, wherein the first plurality ofoligonucleotides comprises a plurality of first capture probes and aplurality of first amplification primers, wherein the first captureprobes are immobilized on the first flow cell at a density lower thanthe first amplification primers, (ii) selectively hybridizing the firsttemplate nucleic acids to the first capture probes, (iii) extending thefirst capture probes hybridized to the first template nucleic acids toobtain first extended polynucleotides, and (iv) removing the firsttemplate nucleic acids from the first extended polynucleotides, and (v)amplifying the first extended polynucleotides by hybridizing the firstextended polynucleotides to the first amplification primers to obtainthe amplified first template nucleic acids; and (b) performing a secondselective amplification on a second flow cell with second templatenucleic acids to obtain amplified second template nucleic acids,comprising; (i) obtaining the second flow cell comprising a secondplurality of oligonucleotides immobilized thereon, wherein the secondplurality of oligonucleotides comprises a plurality of second captureprobes and a plurality of second amplification primers, wherein thesecond capture probes are immobilized on the second flow cell at adensity lower than the second amplification primers, and wherein anamount of the second capture probes immobilized on the second flow cellis substantially the same as an amount of the first capture probesimmobilized on the first flow cell, (ii) selectively hybridizing thesecond template nucleic acids to the second capture probes, (iii)extending the second capture probes hybridized to the second templatenucleic acids to obtain second extended polynucleotides, (iv) removingthe second template nucleic acids from the second extendedpolynucleotides, and (v) amplifying the second extended polynucleotidesby hybridizing the second extended polynucleotides to the secondamplification primers to obtain the amplified second template nucleicacids; wherein an amount of the amplified first template nucleic acidsis substantially the same as an amount of the amplified second templatenucleic acids.
 40. The method of claim 39, wherein the first and secondamplification primers are blocked prior to the extending steps; andwherein the first and second amplification primers are deblocked priorto the amplifying steps.